AperTO - Archivio Istituzionale Open Access dell'Università di Torino Boletus mendax, a new species of Boletus sect. Luridi from Italy and insights on the B. luridus complex This is the author's manuscript Original Citation: Availability: This version is available http://hdl.handle.net/2318/132875 since Published version: DOI:10.1007/s11557-013-0896-4 Terms of use: Open Access Anyone can freely access the full text of works made available as "Open Access". Works made available under a Creative Commons license can be used according to the terms and conditions of said license. Use of all other works requires consent of the right holder (author or publisher) if not exempted from copyright protection by the applicable law. (Article begins on next page) 04 October 2021 This is an author version of the contribution published on: A. Vizzini; G. Simonini; E. Ercole; S. Voyron (2014) Boletus mendax, a new species of Boletus sect. Luridi from Italy and insights on the B. luridus complex, MYCOLOGICAL PROGRESS (ISSN:1617-416X), pp. 95- 109. Vol. 13. 10.1007/s11557-013-0896-4 The definitive version is available at: http://link.springer.com/article/10.1007%2Fs11557-013-0896-4 Boletus mendax, a new species of Boletus sect. Luridi from Italy and insights on the B. luridus complex Alfredo Vizzini , Giampaolo Simonini, Enrico Ercole and Samuele Voyron Abstract The new species Boletus mendax of sect. Luridi is reported and discussed; its morphological, anatomical and edaphic characters are described and a molecular investigation is carried out in order to elucidate the separation from neighboring taxa. The present research also demonstrates the high degree of segregation of two collections of the recently described Boletus comptus and the low taxonomic value of the usual reddish pigmentation of the subhymenophoral layer (the so called Bataille’s line) for both taxa B. mendax and B. luridus. Colour pictures taken in habitat of the taxa presented herein are also provided. Finally, according to an ITS analysis, sect. Luridi is shown to be polyphyletic and the importance of morphological features as stipe ornamentation, context colour beneath tubes and amyloid reaction in stipe base is discussed. Introduction Section Luridi Fr. ex Lannoy & Estades, typified by B. luridus Schaeff., is a traditionally well-defined group in the genus Boletus L. (Boletaceae Chevall.), distributed mainly in temperate Europe (Singer 1967; Pilát and Dermek 1974; Engel et al. 1983; Alessio 1985; Lannoy and Estades 2001; Muñoz 2005; Klofac 2007; Šutara et al. 2009; Knudsen and Taylor 2012) and North/Central America (Coker and Beers 1943; Singer 1947; Snell and Dick 1970; Smith and Thiers 1971; Thiers 1975; Grund and Harrison 1976; Both 1993; Bessette et al. 1997, 2000; Ortiz-Santana et al. 2007), as well as temperate eastern and tropical southeastern Asia (Corner 1972; Zang 2006; Takahashi et al. 2011) and Australia (Watling and Li 1999). The species of this section are characterized by a boletoid habit, small, often orange-reddish pores, a reticulate or minutely furfuraceous stipe surface and usually blueing tubes, pores and context. According to a recent preliminary molecular analysis on Mediterranean Boletus (Marques et al. 2010), sect. Luridi is polyphyletic and split into three clades. Within this section, Boletus luridus is distinguished in having a pileus variable in colour, olivaceous to snuff-brown, peach or brick-coloured to reddish brown, pores orange-red even when young, context red to blood-red at the base of tubes (due to the Bataille’s line), stipe cylindric-clavate usually not robust and markedly swollen, coarsely and strongly reticulate with a red network overall and an amyloid reaction on basal stipe context (Singer 1967; Leclair and Essette 1969; Watling 1970; Pilát and Dermek 1974; Engel et al. 1983; Alessio 1985; Lannoy and Estades 2001; Muñoz 2005; Watling and Hills 2005; Galli 2007; Klofac 2007; Šutara et al. 2009; Kibby 2011; Knudsen and Taylor 2012). The variability of B. luridus is puzzling: based only on morphological data it is hard to define whether, as currently delimited, it is a single, very variable taxon, or a collective species (Simonini 2001). In the latter case, what are the characters useful for their differentiation and at what taxonomic rank should we place the neighbouring taxa? 1 As it is well known, B. luridus displays a reliable chromatic character: the so-called Bataille’s line (Lannoy and Estades 2001; Muñoz 2005), probably observed by Bataille (1908) for the first time, i.e., a dull red coloured thin layer between tubes and pileus context. Such a feature is not present in any other European and extra European species belonging to section Luridi, even those considered most similar and taxonomically allied to B. luridus. The real origin of the Bataille’s line is to date, unknown, and the constancy of this character is somewhat questionable. Moreover, the opposite (and not necessarily antithetical) statement is also questionable: is it possible that more taxa can have the Bataille’s line? Our work aims to consider a new taxon having the Bataille’s line (at least in the majority of cases) like B. luridus, but different from this latter species in the general appearance, colours, size of the spores, pileipellis structure and habitat. In addition, the monophyly of section Luridi and its morphologically based splitting in subsections and stirps (Lannoy and Estades 2001; Klofac 2007) are tested against the molecular phylogenetic approach. Materials and methods Macro- and microscopic features Fresh basidiomata were photographed in the field to show their distinctive characters before being collected; notes on accompanying vegetation and on soil type and characteristics were taken. A Pentax K5 reflex digital camera with lens Pentax SMC D FA Macro WR 1:1 100 mm was used. Photographs were taken in day light or with flash Pentax AF 360 FGZ light. The colour of spore print was estimated the day after the deposit, in dry conditions. All the colour descriptions were based on Kornerup and Wanscher (1978). Microscopic features were observed on well-dried material; free-hand sections of dried material were revived in L4 solution, Melzer’s reagent (for spores) or in ammoniacal Congo red (pileipellis, hymenium). Measurements were made at 1000 × magnification, with an ocular micrometer. Spore dimensions were taken from the pileus hymenophore of mature specimens, randomly chosen, and given as: (minimum–) average minus standard deviation – average plus standard deviation (– maximum) of length × (minimum–) average minus standard deviation – average plus standard deviation (–maximum) of width [μm]; Q = (minimum–) average minus standard deviation – average plus standard deviation (–maximum) of ratio length/width, Qm = average ± standard deviation of ratio length/width, V = (minimum–) average minus standard deviation – average plus standard deviation (–maximum) of the volume [μm3], Vm = average ± standard deviation of the volume [μm3]. The approximate, conventional spore volume was estimated as a rotation ellipsoid with the formula: V = 4/3*(length/2)*((width/2)*width) *π/2. To assess spore size, at least 31 spores per collection were measured. The notation (c = X) means that measurements were made on X collections; standard deviation, maximum and minimum are expressed with reference to the X average values. After measurement of dimensions, spores were further tested for amylodity in Melzer’s reagent. Anatomical microscopical features were observed on glass slides with a Jenamed Variant optical light microscope, and all line-drawings of microstructures were made from rehydrated material. Photographs are taken from the microscope through a Jena phototube extension. Amyloid reaction of the context with Melzer’s reagent was tested in the stipe base following Imler’s procedure (Imler 1950; Singer 1965). Specimens of the type collection examined in this study were deposited in AMB (isotype in GS). Herbarium acronyms follow Thiers (2013) except “GS” that refers to the personal herbarium of 2 Giampaolo Simonini. Author citations follow the Index Fungorum-Authors of Fungal Names (http://www.indexfungorum.org/authorsoffungalnames.htm). The infra-generic (sectional) taxonomic classification of Boletus follows Muñoz (2005). DNA extraction, PCR amplification, and DNA sequencing Genomic DNA was isolated from 1 mg of 10 herbarium specimens (in succession from AMB12632 to AMB12641) using the DNeasy Plant Mini Kit (Qiagen, Milan Italy). Universal primers ITS1F/ITS4 were used for the ITS region amplification (White et al. 1990; Gardes and Bruns 1993). Amplification reactions were performed in PE9700 thermal cycler (Perkin-Elmer, Applied Biosystems) in a 25 μl reaction mixture using the following final concentrations or total amounts: 5 ng DNA, 1 × PCR buffer (20 mM Tris/HCl pH 8.4, 50 mM KCl), 1 μM of each primer, 2.5 mM MgCl2, 0.25 mM of each dNTP, 0.5 unit of Taq polymerase (Promega). The PCR program was as follows: 3 min at 95 °C for 1 cycle; 30 s at 94 °C, 45 s at 50 °C, 2 min at 72 °C for 35 cycles, 10 min at 72 °C for 1 cycle. PCR products were resolved on a 1.0 % agarose gel and visualized by staining with ethidium bromide. PCR products were purified and sequenced by MACROGEN Inc. (Seoul, Republic of Korea). Sequence assembly and editing were performed using Geneious v5.3 (Drummond et al. 2010). Sequences were deposited in GenBank under the accession numbers given in Table 1. Table 1 Boletus sequences newly generated for this study and associated GenBank accession numbers ITS GenBank Species Collection data Voucher Herbarium n. acc. n. B. 24.08.10, Agriturismo Rio Riccò, Busana (RE), Castanea AMB12632 KC734547 mendax 1 sativa, leg. G. Simonini B. 27.08.10, Rifugio Zamboni, Villa Minozzo (RE), Fagus AMB12633 KC734548 mendax 2 sylvatica, leg. G. Simonini (Holotype) B. 24.08.10, Il Castagno, Busana (RE), Castanea sativa, AMB12634 KC734543 mendax 3 leg. G. Simonini B. 28.08.10, Agriturismo Rio Riccò, Busana (RE), Castanea AMB12635 KC734545 mendax 4 sativa, leg.